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INTRODUCTION

Kidney is the main metabolic and excretory organ of

drugs, and is also the main target of drug toxicity. It has

been reported that 34.2% of acute renal failure and 20 %

of end-stage kidney disease are caused by side effects of

drugs, respectively [1]. Cisplatin (Cp) is an inorganic

platinum-based chemotherapy drug. At present, Cp is

widely used in the treatment of various malignant

tumors, including malignant tumors of the head and

neck, lung cancer, ovarian cancer, testicular cancer,

bladder cancer [2], and its efficacy is proportional to the

dose [3]. However, limiting its full clinical potential is

that it has various significant side effects, such as

myelosuppression, peripheral neuropathy, ototoxicity,

allergic reactions, nephrotoxicity, etc., of which

nephrotoxicity is its most serious side effect [4].

Clinically, a single dose of cisplatin (50~100 mg/m2)

causes the incidence of nephrotoxicity to be as high as

1/3 [5]. Therefore, the prevention and treatment of

nephrotoxicity during chemotherapy is one of the urgent

problems to be solved in clinical practice.

Mesenchymal stem cells (MSCs) are pluripotent stem

cells derived from mesoderm and have the ability to

multi-directionally differentiate into fat, osteogenesis,

cartilage and neuron, which are present in various

tissues and organs of the body [6]. MSCs can homing to

the injury site in vivo and promote tissue damage repair

by differentiation into damaged cells and paracrine

www.aging-us.com AGING 2020, Vol. 12, No. 18

Research Paper

Pre-incubation with human umbilical cord derived mesenchymal stem cells-exosomes prevents cisplatin-induced renal tubular epithelial cell injury

Zongying Li1, Shuyi Cao1 1Department of Nephrology, Cangzhou Central Hospital, Cangzhou, Hebei Province, China

Correspondence to: Zongying Li; email: linjieku8297@163.com; https://orcid.org/0000-0002-0163-9928 Keywords: apoptosis, cisplatin, exosomes, renal tubular epithelial cell, viability nephrotoxicity Received: February 29, 2020 Accepted: June 4, 2020 Published: September 23, 2020

Copyright: © 2020 Li and Cao. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

Purpose: The administration of cisplatin is limited due to its nephrotoxicity, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. Results: In vitro, the NRK cells pre-incubated with HUMSC-exosomes increased the Cp-inhibited cell viability, proliferation activity, and the cell proportion in G1-phase and inhibited Cp-induced cell apoptosis. Furthermore, the expression levels of apoptotic marker proteins Bim, Bad, Bax, cleaved caspase-3, and cleaved caspase-9 induced by Cp in the NRK cells were decreased by pre-incubating with HUMSC-exosomes. Conclusion: Our findings indicated that the exosomes from HUMSCs can effectively increase the survival rate and inhibit cell apoptosis of NRK cells. Therefore, pre-treatment of HUMSC-exosomes may be a new method to improve the therapeutic effect of cisplatin. Patients and methods: Exosomes were isolated from human umbilical cord derived mesenchymal stem cells (HUMSCs). Co-culture of normal rat renal tubular epithelial cells (NRK) and the absorption of exogenous exosomes by NRK cells were examined in vitro. Then the NRK cells were incubated with exosomes from HUMSCs and cisplatin (Cp). Cells were harvested for MTT assay, cloning formation, flow cytometry, and Western blot.

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pathways [7]. As an important substance in the

secretion of paracrine cells, exosomes play a significant

role in promoting cartilage regeneration, reducing

ischemia-reperfusion injury and liver and kidney

damage [8–11]. Exosomes are biologically active

extracellular vesicles secreted by living cells, ranging in

size from 30 to 200 nm, which contain abundant

biologically active substances such as miRNA, mRNA,

and protein [12]. Therefore, exosomes can participate in

the regulation of many life activities, and play a greater

role in information transmission, disease diagnosis and

treatment [13, 14]. Studies have shown that exosomes

can effectively reduce the nephrotoxicity of glycerol,

gentamicin and cyclosporine by inhibiting oxidative

stress [15, 16]. The research implies that exosomes from

MSCs may be a novel stem cell-based therapy for

kidney diseases.

Therefore, exosomes may be able to reduce Cp-induced

kidney damage. In this work, we selected HUMSC-

derived exosomes, which confirmed that it can protect

and prevent Cp-induced kidney damage. Furthermore,

we investigated the protective mechanism of Cp-induced

renal tubular cytotoxicity by exosomes, and provided

experimental evidence for further clinical research.

RESULTS

Characterization and identification of exosomes

derived from HUMSCs

The particle size parameters of the exosomes were

measured by TEM and DLS. The results showed that

the exosomes were microvesicles with a diameter of 80

to 110 nm (Figure 1A), and the peak size of the particle

Figure 1. Identification of exosomes from Human umbilical cord derived mesenchymal stem cells (HUMSCs). (A) Observation of the shape and size of exosomes by transmission electron microscopy. (B) Measurement of the size of exosomes by dynamic light scattering. (C) Zeta potential of the exosomes. (D) The expression of CD9 and CD63, the surface markers of exosomes by Western blot.

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size distribution was 103 nm (Figure 1B). The zeta

potential of the exosomes was -24.15±3.1 mV (Figure

1C). Western blot results showed that the exosomes

surface markers CD9 and CD63 were highly expressed

(Figure 1D). These results suggest that HUMSC-

exosomes were successfully separated.

The exosomes were taken up by NRK cells

NRK cells and the exosomes were co-cultured for 6 h.

The confocal microscopy observation results showed

that Dil-labelled exosomes (red) were taken up by NRK

cells, successfully (Figure 2A, 2B).

Exosomes protect against the injury of NRK cells

induced by Cp

The viability of NRK cells co-cultured with exosomes were

tested after treating with 5 μM, 10 μM, 20 μM, 40 μM, 80

μM, and 160 μM of Cp, respectively. The MTT results

illustrated that NRK cells co-cultured with exosomes for

12 h and 24 h facilitated the viability, while which was

inhibited by 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM

of Cp (Figure 3A). The colony formation of NRK cells

was tested after treating with Cp, Exo1h+Cp, Exo6h+Cp,

Exo12h+Cp, and Exo24h+Cp, respectively. Compared

with the Control group, the number of cell colony in the

Cp group was decreased. However, relative to the Cp

group, the number of cell colony was upregulated in the

Exo6h+Cp, Exo12h+Cp, and Exo24h+Cp groups (Figure

3B, 3C), indicating that HUMSC-exosomes relieved Cp-

induced injury of NRK cells.

Exosomes attenuates Cp induced NRK cells

apoptosis

The apoptosis of NRK cells was tested after treating

with Exo1h+Cp, Exo6h+Cp, Exo12h+Cp, and

Exo24h+Cp. Compared with the Control group, the

apoptosis levels of the Cp group were upregulated

(Figure 4A). However, relative to the Cp group, the

apoptosis levels were downregulated in the Exo1h+Cp,

Exo6h+Cp, Exo12h+Cp, and Exo24h+Cp groups

(Figure 4A).

Figure 2. He exosomes were taken up by NRK. (A) After NRK co-cultured with exosomes, the location of Dil (red), the marker of exosomes, and nucleus (blue) were observed by confocal microscopy. (B) The changes of fluorescence intensity of Dil were analyzed by ImageJ.

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Exosomes regulates the expression of apoptotic

marker proteins in NRK cells treated with Cp

To further clarify the protective mechanism of

HUMSC-exosomes on apoptosis of kidney cells, the

expression levels of apoptotic marker proteins Bax, Bid,

Bim, Bcl-2, cleaved caspase-3, and cleaved caspase-9

were measured by Western blot. Compared with the

Control group, the level of Bcl-2 was decreased and the

levels of Bax, Bid, Bim, cleaved caspase-3, and cleaved

caspase-9 were upregulated in the Cp group (Figure

4B). However, relative to the Cp group, the levels of

Bcl-2 were upregulated and the levels of Bax, Bid, Bim,

cleaved caspase-3, and cleaved caspase-9 were

downregulated in the Cp group in the Exo1h+Cp,

Exo6h+Cp, Exo12h+Cp, and Exo24h+Cp groups

(Figure 4B). It was illustrated that HUMSC-exosomes

inhibited the NRK cells apoptosis induced by Cp.

Exosomes regulates the cell cycle progression of

NRK cells

The changes of cell cycle progression were measured

after treating with Exo1h+Cp, Exo6h+Cp, Exo12h+Cp,

and Exo24h+Cp. Compared with the Control group, the

proportion of the cells in G1-phase in the Cp group was

decreased (Figure 5) and that of the cells in the G2-phase

was increased (Figure 5). However, relative to the Cp

group, the proportion of the cells in G1-phase in the

Exo24h+Cp group was increased (Figure 5), suggesting

that HUMSC-exosomes could improve cycle progression

of NRK cells.

Figure 3. Cell viability and colony formation detection. (A) After NRK co-cultured with exosomes and Cp, the percentage of cell viability was measured by MTT assay; *, p < 0.05, **, p < 0.01, ***, p < 0.001. (B) and (C) The changes of colony number were measured by colony formation assay. *, p < 0.05 vs. Control; #, p < 0.05 vs. Exo1h+Cp; &, p < 0.05 vs. Exo6h+Cp; $, p < 0.05 vs. Exo12h+Cp.

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DISCUSSION

Cp is a potent anticancer agent and its mechanism of

action may be related to its high intracellular reactants.

In an aqueous environment in a cell, Cp forms a

conjugate with intracellular glutathione, protein, RNA,

and DNA, and crosslinks with the purine base of DNA,

interfering with DNA repair mechanisms, leading to

DNA damage, further induction. Thus, it induces cancer

cells to be apoptotic, but they are also toxic to normal

cells [17]. Cp and its hydrated or hydroxyl metabolites

are excreted primarily through the kidneys. Because of

low molecular weight and no charge of Cp, the free Cp

in plasma is easily filtered by the glomerulus, which

results in a concentration of 5 times that in the proximal

tubular epithelial cells. It is consequently more sensitive

to the toxic effects of Cp, and the nephrotoxicity of Cp

is most serious. However, the nephrotoxicity caused by

Cp is currently lacking effective prevention and

treatment methods [18]. In the present study, Cp with

gradually elevated concentration decreased the viability

and colony formation of the NRK with dose-dependent.

It was indicated that Cp was highly toxic to the NRK.

Furthermore, studies have shown that Cp damages

proximal tubular epithelial cells, leading to apoptosis of

renal tubular cells mainly involved in mitochondria-

mediated endogenous pathways, death receptor-

mediated exogenous pathways and endoplasmic

reticulum stress pathways [19]. We also verified the

induction effect of Cp on NRK apoptosis in the present

study, which was consistent with previous research

results.

Figure 4. Effect of different groups on NRK apoptosis. (A) Apoptosis was tested by Annexin V-FITC/PI staining method. (B) The protein levels of Bax, Bid, Bim, Bcl-2, cleaved caspase-3, and cleaved caspase-9 were measured by Western blot, and analyzed by ImageJ. * P< 0.05 vs. Control; &, p < 0.05 vs. Exo1h+Cp.

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Figure 5. Effect of different groups on NRK cell cycle progression. The cell cycle progression was tested by flow cytometry.

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In recent years, as the main biological component of

paracrine secretion of stem cells, exosomes reduce the

possibility of stem cells forming tumors in vivo because

they do not have the ability to divide and differentiate

[20, 21]. Compared with mesenchymal stem cell

transplantation therapy, exosomes have relative

stability. Exosomes are distinguished by their size from

other vesicles secreted by mesenchymal stem cells,

ranging from 30-200 nm in diameter to densities

ranging from 1.10 to 1.20 g/mL [12]. The key step in

the present study is the extraction of exosomes. The

present study used the low temperature ultra-high speed

centrifugation method to obtain relatively pure

exosomes. Under electron microscope, the exosomes

were mostly concentrated at 80-110 nm, and can

express CD9 and CD63 marker surface proteins.

Consistent with the biological characteristics and

identification criteria of exosomes, it indicated that the

exosomes of HUMSCs were successfully isolated.

Furthermore, in pig and mouse models of myocardial

ischemia-reperfusion injury in mice, Timmers et al. [22]

found that the medium reduced myocardial infarct size

by 60% and 50%, respectively, when intravenously

injected into conditioned medium of mesenchymal stem

cells. It was further confirmed that the size of the active

medium acting was in the range of 50-200 nm. The

study found that mesenchymal stem cell conditioned

medium can reduce myocardial infarct size in mice, but

conditioned medium without exosomes does not have

this function [23]. All of the above studies have

demonstrated that mesenchymal stem cells mainly

function through exosomes in their supernatants. In the

present study, notably, when exosomes were co-

cultured with Cp-treated NRK, the cell viability of NRK

was remarkably higher than that of the Cp-treatment

alone. In addition, by increasing the culture time of

exosomes and NRK, it was found that the proliferative

capacity of NRK was positively correlated with the

culture time of exosomes. These results suggested that

exosomes from HUMSCs promoted renal endothelial

cell proliferation.

In order to explore the protection of the exosomes on

Cp-induced NRK cell injury, its effect on apoptosis

was observed. The results showed that the apoptosis

rate of NRK cells was up-regulated after Cp treatment,

and the expression of apoptosis markers Bax, Bid, Bim,

Caspas-3 and -9 were up-regulated. Notably, after the

exosomes were added to the Cp-induced NR, the

apoptotic rate increased significantly, and the

expressions of Bax, Bid, Bim, and Caspas-3 and -9

were all down-regulated, and the expression of Bcl-2

was upregulated. It was indicated that exosomes

promoted the proliferation of NRK cells and inhibit

the apoptosis of cells, which consistent with previous

results in exosomes regulating apoptosis and

proliferation of endothelial cells [24]. Our further

results showed that the cells incubated with exosomes

alleviated cell cycle inhibition caused by Cp. In recent

years, a large number of studies have shown that

exosome miRNAs play an important role in the

occurrence and development of diseases. Exosomes can

selectively encapsulate miRNAs and stably transfer

miRNAs to recipient cells and function [25].

Additionally, in diseases such as lung cancer, lung

inflammation, and pulmonary fibrosis, exosome

miRNAs regulate the expression of many proliferation-

related and/or apoptosis-related genes [25–27]. For

example, cardiac stem cell-derived exosomes miR-21

inhibited cardiomyocyte apoptosis by targeting binding

to programmed cell death factor 4 [28]. We

hypothesized that the protective effect of exosomes on

NRK cells may be related to the regulation of cell

proliferation and apoptosis-related genes by the

miRNAs they carry. However, this speculation still

needs further experimental verification.

The clinical application of exosomes depends on the

technical breakthrough of exosome-based drug delivery

system. At present, exosomes have been found to play

an important role in various health and disease models

through the transmission of molecular information.

Exosomes are recognized as biomarkers and prognostic

factors of diseases and have important clinical

diagnostic and therapeutic significance. In addition,

they have the potential to be used clinically as a vehicle

for gene and drug delivery. Exosomes themselves are

quite inert, but when they fuse with the cell membrane,

they can deliver the materials and signals they carry to

the recipient cell and change its biological function.

Therefore, exosomes are potential carriers for

nanometer drug delivery or gene therapy. As natural

carriers of functional small RNAs and proteins,

exosomes can be used to deliver various ribonucleic

acid molecules, peptides and synthetic drugs in the field

of drug delivery.

In this study, the functionally exosomal miRNAs have

not been screened and explored. In the later stage, we

will screen for miRNAs in the process of regulating the

function of renal tubular epithelial cells by miRNA

microarray or high throughput sequencing technology.

In summary, exosomes from HUMSCs can significantly

reduce Cp-induced NRK cell injury, which may be

related to the up-regulation of Bcl-2 in renal tubular

cells and inhibition of Bax, Bid, and Bim expressions,

thereby inhibiting downstream caspase-3 and caspase-9

activation. Exosomes may be an ideal Cp nephrotoxicity

control drug and deserve further study. And these

findings provide a basis for the future use of exosomes

as a new biological therapeutic approach for renal

diseases and injuries.

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CONCLUSION

In this study, the functionally exosomal miRNAs have

not been screened and explored. In the later stage, we

will screen for miRNAs in the process of regulating the

function of renal tubular epithelial cells by miRNA

microarray or high throughput sequencing technology.

In summary, exosomes from HUMSCs can significantly

reduce Cp-induced NRK cell injury, which may be

related to the up-regulation of Bcl-2 in renal tubular

cells and inhibition of Bax, Bid, and Bim expressions,

thereby inhibiting downstream caspase-3 and caspase-9

activation. Exosomes may be an ideal Cp nephrotoxicity

control drug and deserve further study. And these

findings provide a basis for the future use of exosomes

as a new biological therapeutic approach for renal

diseases and injuries.

MATERIALS AND METHODS

Cell culture

HUMSCs were purchased from Cyagen Biosciences

(Guangzhou, China). HUMSCs were culture in DMEM

containing 10% fetal bovine serum at 37 °C and in 5%

CO2 incubator. Furthermore, normal rat renal tubular

epithelial cells (NRK) were purchased from Procell

(Wuhan, China) and were cultured in DMEM

(PM150210) containing 10% fetal bovine serum at 37

°C and in 5% CO2 incubator.

Exosomes separation

The culture supernatant of HUMSCs in 4-6 generations

was collected and the exosomes were extracted

according to the instructions of the Invitrogen 4478359

Total Exosome Isolation Reagent (Gibco, Invitrogen,

UK). Briefly, the culture supernatant was transferred to

a high-speed centrifuge tube, centrifuge at 4°C, 2000 g

for 30 min, the supernatant was taken and transferred to

a new high-speed centrifuge tube, and the cell

supernatant was added (cell supernatant: reagent = 2:1).

After fully mixing with vortex, the mixture was placed

in a refrigerator and incubated at 4 °C overnight. After

centrifugation at 4 °C, 10000 g for 60 min, the

precipitate was resuspended in 100 μl of cold PBS as a

suspension of exosomes and stored at -80 °C.

Identification of exosomes

Transmission electron microscope (TEM) was used to

observe the morphology of exosomes. 10 μl of the

exosomes were separated and purified, and diluted with

an equal volume of balanced salt PBS solution. The

sample was added dropwise to a sample-loaded copper

mesh, and was allowed to stand at room temperature for

1 minute. Then the excess liquid was gently removed

using filter paper. After that, the sample was negatively

stained with 3% sodium phosphotungstate solution (pH

6.8) for 5 minutes at RT. After gently washing with

double distilled water, the sample was air-dried at room

temperature, and observed under a transmission electron

microscope. In addition, dynamic light scattering (DLS)

was used to detect the size of exosomes. Briefly, 10 μl

of the exosomes were separated and purified, and

diluted with PBS solution to measure the size using a

Malvern laser particle size analyzer.

Western blot

Western Blot was used to identify the expressions of

CD9 and CD63 on the surface of exosomes and the

expressions of Bax, Bid, Bim, Bcl-2, cleaved caspase-3,

and cleaved caspase-9 in NRK cells. Total proteins

were obtained using lysis buffer, and then quantitated

by bicinchoninic acid kit (Beyotime, Shanghai, China).

Following sample separating and transferring into

PVDF membranes, membranes were immerged in 5%

nonfat milk. Next, primary antibodies of CD9, CD6,

Bax, Bid, Bim, Bcl-2, cleaved caspase-3, cleaved

caspase-9 (1: 800, Abcam), and β-actin (1: 1000,

Beyotime), respectively, were used for immunoblotting

of the membranes overnight at 4°C. After the incubation

with second antibody (1:5000, Jackson, USA), the

protein levels were detected by enhanced

chemiluminescence (ECL, Millipore, USA).

Observation of cell uptake by laser confocal

microscopy

Dil (Thermo Fisher Scientific, Invitrogen, catalog

number: D282) was used to mark the exosomes. After

staining for 30 min, the exosomes were washed with

PBS and collected by centrifugation at 10000 g for 60

min to obtain the Dil-labeled exosomes. NRK cells

were planted in confocal dishes. After the cells were

attached overnight, 20 μg/ml of Dil-labeled exosomes

were added to the dish, and after 1, 4, 8, 12, and 24 h of

culture, the cells were washed with PBS and fixed with

4% paraformaldehyde for 30 min. Subsequently, after

washing with PBS, the cells were permeabilized for 15

min using 0.2% Triton-100. Nucleus were stained with

Hoechst 33342, and photographed using laser confocal

after washing with PBS.

Cell viability by MTT (thiazolyl blue tetrazolium

bromide)

NRK cells were pre-incubated with exosomes from HUMSCs for 1 h, 6 h, 12 h, and 24h, followed by

treatment with Cp at 5, 10, 20, 40, 80, and 160 μM in 96-well plates. After co-culturing 24h, cell viability was

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measured using the MTT assay. MTT solution (5 g/L) was added at 10 μL/well. After incubating for 4 h, the

supernatant was removed, 150 μL of DMSO was added to each well, and the cells were gently shaken for 30 s.

100 μL of each well was aspirated into the enzyme label, and then the absorbance value (OD) at 570 nm

was measured by a microplate reader. The percentage of

cell activity was used as an evaluation index for the effect of nanoparticles on cell viability. The calculation

formula was: percentage of cell activity (%) = (OD value of each well in the infected group - blank OD

value) / (Control group OD mean - blank OD value) ×

100%, control group cell activity percentage was 100%.

Colony formation assay

NRK cells in the Control, Cp, Exo1h+Cp, Exo6h+Cp, Exo12h+Cp, and Exo24h+Cp. groups were respectively

mixed with 0.3% Noble agar in 10% FBS supplemented

DMEM, which were seeded as the upper agar layer of the agar plates. The cells were then incubated in a 37 ˚C

incubator for 7 days, and the number of colonies was counted.

Cell apoptosis by flow cytometry

NRK cells were seeded in 6-well plates overnight, 200 μg/ml of exosomes was added. After co-culturing for 1

h, 6 h, 12 h, and 24 h, 20 μM Cp was added in to the plates. The NRK cells were divided into six groups: no

treatment group (Control), Cp group, co-culture with

exosomes combined with Cp treatment groups for 1 h (Exo1h+Cp), 6 h (Exo6h+Cp), 12 h (Exo12h+Cp), and

24 h (Exo24h+Cp). After Cp treatment for 24 hours, cell apoptosis was detected by flow cytometry using

Annexin-PI staining.

Cell cycle detection by flow cytometry

The NRK cells were seeded in 6-well plates overnight

were pre-incubated with exosomes from HUMSCs for 1 h, 6 h, 12 h, and 24h, followed by treatment with 20 μM

Cp which prepared by a medium containing 200 μg/ml

of exosomes. Then, the cells in each group were collected. 5X104 cells in each group were washed twice

with PBS, and fixed in 1 mL of 70% ice ethanol for 24 h at 4 °C. Then, the cells were washed twice with PBS

and were added with propidium iodide staining solution

at 4 °C for 30 min. Cell cycle distribution was measured using a flow cytometer.

Statistical analysis

GraphPad Prism 6.05 was used for statistical analysis of

experimental data. One-way ANOVA and Student's t

test were used for comparative analysis of differences. P < 0.05 was considered statistically significant.

AUTHOR CONTRIBUTIONS

Zongying Li performed the majority of experiments and

analyzed the data; Shuyi Cao performed the molecular

investigations; Zongying Li designed and coordinated

the research; Shuyi Cao rote the paper.

CONFLICTS OF INTEREST

The author reports no conflicts of interest in this work.

FUNDING

This research did not receive any specific grant from

funding agencies in the public, commercial, or not-for-

profit sectors.

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